Ba2+ has multiple effects on presynaptic terminals. The ion inhibits the K+ channels responsible for stabilizing the plasma membrane potential in the same way as previously reported for dendrotoxin and 4-aminopyridine. Secondly, the ion can substitute fully for Ca2+ in supporting KCl-evoked release of glutamate from guinea-pig cerebrocortical synaptosomes. In the latter case, the kinetics of glutamate release in the presence of saturating Ca2+ or Ba2+ are essentially identical. Substantially lower external concentrations of Ba2+ are required to achieve the same release kinetics as with Ca2+. The average internal free Ba2+ concentration attained during KCl depolarization is some 10-fold higher than that for Ca2+. However, because the fura-2 signal reflects predominantly the overflow of divalent cation after dissociation from the release trigger, it is not the valid parameter to compare effectiveness of the cations in triggering glutamate exocytosis. In view of the established inability of Ba2+ to interact with calmodulin, these results are discussed in relation to theories in which Ca2+/calmodulin-dependent protein kinase-mediated phosphorylation is a prerequisite for synaptic vesicle exocytosis.